The long term goal of this grant will continue to be the identification of molecular mechanisms that are at the basis of specificity in G protein function, especially with reference to the G protein BetaGamma complex. The individual functions of the beta and gamma subunit families in signaling are unclear. The specific aims in the proposal test three different hypotheses: (i) that the G protein gamma subunit plays a role in receptor activation of a G protein; (ii) that members of the gamma subunit family influence specificity of G proteins for receptors and (iii) that the prenyl group which modifies the gamma subunit, interacts with a specific domain on a receptor and an effector. Experiments to address gamma subunit function will be performed in vitro with purified recombinant G proteins and a muscarinic receptor. Mutant alpha and gamma subunits will be used to identify the specific contributions of these two subunits to the multi-step process of G protein activation by the receptor. Specificity between receptors and G protein gamma subunits will first be tested in intact neurons using elctrophysiological assays and gamma subunit specific peptides. The underlying kinetic basis for specificity detected in these assays will be identified in vitro by measuring various steps during the functional interaction of appropriate receptor-G protein combinations. Evidence for direct binding of the prenyl moiety to a receptor, M2 muscarinic and an effector, phospholipase C- beta3 (PLCbeta3-BetaGamma), will be obtained by photoaffinity labeling with novel photoactive analogs of the prenyl moiety. The site on the receptor/effector that binds the prenyl moiety will be identified using this approach. To obtain further evidence for prenyl group binding to an effector and to elucidate the structural basis of BetaGamma complex interaction with an effector, a PLCbeta3-BetaGamma complex will be crystallized in a collaborative effort. Achieving the aims of this proposal can help elucidate the basis of two novel molecular interactions. (i) G protein gamma subunit types with receptors. (ii) The gamma subunit prenyl moiety with receptors and effectors. This along with information about the structural bases of interaction between BetaGamma and PLCbeta can help design agents that will specifically disrupt intracellular signaling pathways. Such agents can be used to dissect signaling pathways experimentally. They can also be of potential therapeutic value in diseases caused by aberrant signaling.